The broad, long-term objective of this proposal is to elucidate the molecular mechanisms governing heme signaling in mammalian cells. Heme plays key regulatory roles in fundamental molecular and cellular processes for systems that sense or use oxygen. In humans, partial defects in heme synthesis lead to a wide variety of diseases including porphyrias and hematological diseases. Therefore, understanding how heme functions is important for human health. The most enigmatic and still poorly understood aspect of heme-related diseases is the neuropathy associated with acute porphyrias. This proposal thus focuses on deciphering heme action in neuronogenesis and in oxygen sensing. Previous studies and our preliminary data suggest that heme plays important roles in neuronogenesis and in oxygen sensing. Therefore, we propose to investigate the molecular mechanisms governing heme action in neuronal signaling and in oxygen sensing. The hypotheses to be tested are (1) heme is critical for NGF-induced neuronal differentiation of PC12 cells, (2) heme selectively promotes neuronal survival of NGF-induced PC12 cells, and (3) in mammalian cells, like in yeast, heme mediates, at least in part, oxygen regulation of gene expression. The Specific Aims are (1) to dissect the molecular mechanism by which heme deficiency prevents NGF-induced neuronal differentiation of PC12 cells, (2) to dissect the molecular mechanism by which heme deficiency causes apoptosis in NGF-induced PC12 cells, and (3) to determine the role of heme in oxygen regulation of gene expression in mammalian cells. Biochemical, biophysical and genetic techniques will be used to dissect heme action in neuronal signaling and transcriptional regulation. These studies will define the role of heme in neuronogenesis and in oxygen sensing and should provide insights into the molecular mechanisms underlying the pathogenesis of heme deficiency-related diseases.